Analysis of samples by mass spectrometry (MS) often involves the use of one or more stages of ion dissociation, generally referred to as tandem MS, MS/MS or MSn analysis. The dissociation of ions generated from a sample yields characteristic product ions, and the measured intensities and mass-to-charge ratios (m/z's) of these product ions is useful for structural elucidation, as well as for detecting and/or quantifying targeted or untargeted analytes with high specificity. Historically, dissociation has been most commonly performed in mass spectrometers by collisionally activated dissociation (CAD often termed collision induced dissociation or CID) techniques which utilize relatively high-energy collisions between precursor ions and a neutral gas such as helium, nitrogen or argon (commonly referred to as collision gases) to generate product ions consisting primarily of the thermodynamically favored fragments, these are known as b- and y-type ions in protein/peptide mass spectrometry and result from the cleavage of the N—C amide bond in the peptide backbone.
While CAD has been successfully employed for analysis of a wide variety of molecules, including biomolecules such as peptides, more recently developed dissociation techniques such as electron transfer dissociation (ETD) have been found to be particularly useful for analysis of intact proteins, especially those with post-translational modifications, among other relatively large molecules and especially larger biomolecules. Another such technique is ultraviolet photodissociation (UVPD), in which analyte precursor ions are irradiated with ultraviolet (UV) radiation produced by a UV source, typically a laser. For protein or polypeptide analytes, absorption of UV radiation causes fragmentation to proceed through all known peptide backbone fragmentation pathways, producing primarily a- and x-type fragment ions, but also b-, c-, y-, and z-type fragment ions as well as side-chain fragment ions. The principles and usage of UVPD are described by Brodbelt et al. (Journal of the American Chemical Society, (2013), 135(34), pp. 12646-12651) and by Reilly et al. (U.S. Pat. No. 7,618,806B2). Generally the terms protein and peptide both indicate polymers of varying lengths of amino acid polymers with proteins generally having a greater number of amino acids than the peptides. The term polypeptide as used herein may mean protein or peptide and is generally used to indicate an amino acid polymer that may be seen as a large peptide or a small protein. Herein the terms protein, polypeptide and peptide may be used interchangeably to describe any length of amino acid polymers.
The term ion trap as used herein means a RF electric field ion containment device where ions may be contained in three dimensions (not just in two dimensions as in the case of an ion guide) and may be a linear two dimensional (2D) ion trap or 3D Paul trap. A linear ion trap may be segmented into a plurality of sections, each section having a separate set of electrodes, for example, a linear ion trap with three discreet ion containment sections may have a front section, a middle section and a rear section. In such a device ions may be contained in the trap in various ways, for example, positive ions may be contained in one or more sections and at the same time, negative ions may be contained in different section(s) of the trap. This feature of a segmented linear ion trap greatly facilitates ion-ion reactions such as proton transfer reactions (PTR). A linear ion trap may comprise two distinct linear ion traps such as a high pressure ion trap and a low pressure ion trap (as used in the Thermo Velos or Fusion lines of mass spectrometers, Thermo Fisher Scientific, San Jose, Calif.).
Ultraviolet photodissociation (UVPD) is a technique that utilizes ultraviolet (UV) light rays from UV emitting lasers with the basic process resulting in fragmentation of precursor ions and the subsequent generation of product ions. Polypeptide sequence determination is central to study of biomolecules and for advancing the field of proteomics and clinical diagnostics. Interrogation of polypeptide sequences is most efficiently probed via mass spectrometry. Modern mass spectrometers come equipped with a large array of fragmentation techniques to enable the study of a broad range of molecular compounds. Fragmentation of intact polypeptide species with the appropriate techniques allows for predictable peptide backbone fragmentation. UVPD provides broad and deep fragmentation of proteins, thus giving high sequence coverage, and is well-suited for high throughput proteomics. It is a promising technique for polypeptide fragmentation that is relatively indiscriminate in its fragmentation products. In this technique, gas phase intact protein ions are irradiated with a UV light source (typically a laser). When using a laser of relatively high photon density and energy, photodissociation may proceed via single photon mechanisms or may proceed by 2 or 3 or more photon mechanisms. With such techniques, fragmentation may proceed through all known peptide backbone fragmentation pathways simultaneously producing a, b, c, x, y, and z fragment ions, and may also include side chain fragmentation.
UVPD fragmentation spectra of intact proteins are complex with many overlapping multiply charged fragment ions distributed over a relatively narrow mass to charge (m/z) range making confident peak assignment difficult, hence, there is a need to simplify such spectra. The most common reaction in MS/MS is that of dissociation following ion activation. In the simplest case, singly charged parent ions fragment to yield singly charged and neutral products. Reactions involving a change in charge, often referred to collectively as charge permutation reactions have been known since the beginning of mass spectrometry. One particular advantage of UVPD is that not only does it inherently provide a broad range of protein/peptide fragmentation coverage but it is suitable in many cases for the identification of post-translational modifications (PTMs) since weakly bonded PTMs such as phosphates or glycans often survive this fragmentation process. The detection of PTMs is of increasing importance in proteomics.
As outlined above, broad fragmentation coverage is a clear advantage of UVPD fragmentation. However, this broad coverage often results in a relatively large number of overlapping higher charge states product ion peak envelopes that become increasingly difficult to deconvolute with increasing molecular weight of the fragment ions. Accordingly, advances in the simplification of UVPD mass spectra are desirable.